Abstract:

A method to treat solid tumors and other oncological diseases consists of
parenterally injecting an agent which destroy's blood's extracellular DNA
into the systemic blood circulation of a cancer patient to slow down
malignant. The agent is embodied in the form of a DNAse enzyme and, more
particularly, as a bovine pancreatic DNAse. Doses from 50,000-250,000,000
Kunz units/day are injected for 5-360 days. A binding agent or an agent
that modifies the chemical composition of the blood extracellular DNA is
additionally injected into the blood. This modifying agent is preferably
an enzyme-ribonuclease.

Claims:

1. A method of treatment for a selected cancer, wherein:said selected
cancer is selected from the group comprising: breast cancer; gastric
cancer; colon cancer; kidney cancer; pancreatic cancer; and malignant
melanoma; andsaid method comprising paternally administering to a
parenterally administering to a patient in need thereof DNAse in doses
and regiments which provide blood plasma DNA-Hydrolytic activity,
measured in blood plasma, to exceed 150 Kunitz units per liter of plasma
during more than 12 hours in total within 24 hours.

2. The Method according to claim 1, wherein said doses of said DNAse are
administered to the patient according to a regime schedule which is
carried out continuously for no less than 48 hours.

3. The method according to claim 1, wherein the DNAse is bovine pancreatic
DNAse I, and the dose is from 50,000 Kunitz units to 250,000,000 Kunitz
units a day for 5-360 days.

4. The method according to claim 1, wherein the DNAse is human recombinant
DNAse.

5. The method according to claim 4, wherein she human recombinant DNAse is
administered at a dose of 0.15 mg/kg -500 mg/kg of body weight daily for
5-360 days.

6. A method according to claim 1 wherein the administering is carried out
for the remaining term of the patient's life.

7. A method according to claim 1, further comprising parenterally
administering to said patients anti-DNA antibodies.

8. A method according to claim 1, further comprising parenterally
administering to said patients a ribonuclease enzyme.

9. A method according to claim 8, further comprising parenterrally
administering to said patient a ribonuclease of Serratia Mercenses
bacterium.

Description:

RELATED APPLICATIONS

[0001]The present invention is a Continuation in Part of U.S. Ser. No.
10/564861, filed on Jan. 12, 2006 and was a National Phase Filing of
PCT/RU2004/00261. Additionally, the following copending applications are
related to the present subject matter: [0002]1. U.S. Ser. No.
10/564,615 for "Method for treating oncological, virulent and somatic
diseases, method for controlling treatment efficiency, pharmaceutical
agents and compositions for carrying out said treatment", filed on Jan.
12, 2006, which was a National Phase Filing of PCT/RU2003/000304;
[0003]2. U.S. Ser. No. 10/564,609 for "Method for Treating Diseases
Associated with Changes of Qualitative and Quantitative Composition of
Blood Extracellular DNA", filed on Jan. 12, 2006, which was a National
Phase Filing of PCT/RU2004/00260; and [0004]3. U.S. Ser. No. 11/919,141
for "Method for retarding unhealthy manifestations brought by ageing of
human beings", filed on Oct. 23, 2007, which was a National Phase Filing
of PCT/RU2005/000236. [0005]The contents of the '861 application are
incorporated by reference herein as if rewritten in their entirety.

BACKGROUND OF THE INVENTION

[0006]1. Field of the Invention

[0007]The present invention generally relates to a method for treating
oncological diseases by administering an agent that destroys
extracellular DNA in the blood of a cancer patient.

[0008]2. Description of the Background Art

[0009]Populations of tumor cells developing in patients have a very high
genetic variability which exceeds a same for healthy cells. Genetic
variability of cancer cell populations causes mutated cells to generate
phenotypes that (1) are insensitive to immune and morphogenetic control,
(2) have an ability to invade and metastasize, and (3) are desensitized
to cancer therapies. Selection and clonal expansion of cancer cells are
both considered to underlie a biological and a clinical progression of
tumors. For this reason, an approach of modern cancer therapies is based
on a destruction of cancer cell clones in patients by means of
chemotherapy, immunotherapy, biotherapy, surgical methods, or a
combination thereof.

[0010]Chemotherapy, radiotherapy, biotherapy and more recent immunotherapy
are the most commonly used non-surgical methods of treating cancer
diseases. These therapies are administered to destruct, to damage or to
inactivate a cancer cell's intracellular DNA.

[0011]The chemotherapy approach is based an administration of well known
compounds: platinum preparations, antracycline antibiotics, alkylating
agents and podophyllotoxins. The radioimmunotherapy approach is based on
irradiation of intracellular DNA of cancer cells' nuclei. Alpha particles
from alpha emitters are specially delivered into the cancerous cells to
increase effects on those cells' intracellular DNA. Biotherapeutic and
immunotherapeutic approaches are based on an induction of apoptosis of
cancer cells, which induces death of the cancer cell. Apoptosis starts
with an activation of intracellular nucleuses and follows with a
degradation of the tumor cell's intracellular DNA. This process is
accomplished, for example, by means of administering genotherapeutic
constructions that consist of genes that induce apoptosis or genes coding
the factors which activate the nucleuses.

[0012]Aguilera. et al. discloses in U.S. Pat. No. 6,455,250 endonuclease
Endo SR to treat cancer diseases by mode of its intracellular delivery
into target cells. This method and chemotherapy, with
Etopozide-4-Demetilpipodophylotoxe (4,6-O-R)-etiliden-b-D-glycopiranozid,
were both selected for a prototype of the present invention.

[0013]Topoizomeraze II is an essential cell enzyme that regulates many
aspects of DNA function. The enzyme is responsible for interconversion of
different topological forms of intracellular DNA by means of a generation
of transitory breaks of double-stranded DNA. Etopozide, as a
Topoizomeraze II inhibitor, increases an intracellular level of "broken
DNA-Topoizomeraze II" complexes.

[0014]The result of this drug's influence is an accumulation of
double-stranded intracellular DNA breaks which lead to the cell's death.
A drawback of this method prototype, along with well-known methods, is
their low efficacy. These methods imply that mostly the cancer cells'
intracellular DNA is the therapeutic target. Because of high genetic
variability, these cancer cells become desensitized to the therapies
before they are adequately eliminated. A further disadvantage is that the
intracellular DNA is a difficult-to-approach target; it leads to
necessary high-dosing antineoplastic chemotherapy and/or other
complicated delivery systems. A final disadvantage to these methods is
that they are highly toxic: their influence on cancerous cells'
intracellular DNA also damages healthy cells' DNA.

SUMMARY OF THE INVENTION

[0015]An object of this invention is to develop a highly efficient cancer
therapy having low toxicity. It is an object to resolve the foregoing
drawbacks by administering into systemic circulation an agent which
destroys blood extracellular DNA.

[0016]The agent is introduced in doses that alter an electrophoretic
profile of blood extracellular DNA, which could be detectable by
pulse-electrophoresis. Doses of the agent are introduced according to a
regime schedule that provides for plasma hydrolytic activity exceeding
150 Kuntz units/liter of blood plasma. This level can be supported for
more than 12 hours within a 24 hour period. The treatment is carried out
continuously for no less than 48 hours. In particular, bovine pancreatic
deoxyribonuclease (DNase) can be introduced parenterally in doses ranging
from 50,000 Kunitz per day to 250,000,000 per day. DNase is an enzyme
that catalyzes the hydrolytic cleavage of phosphodiester linkages in the
DNA backbone. These doses of DNase are administered anywhere between five
and 360 days. In particular, recombinant human Dnase (domase-alpha) can
be parenterally introduced in doses ranging from 0.15 mg/day to 500
mg/day between a five-360 day period. The treatment may continue for a
life of the patient. Additionally, an agent which bounds extracellular
DNA, s.a., anti-DNA antibodies, can also be introduced to the systemic
circulation. A modifying agent can further be introduced into the
circulation, which modifies the chemical structure, the conformation, the
degree of polymerization, or the association of proteins, lipids and/or
ribonucleic acids of the blood's extracellular DNA. A preferred modifying
agent may be a ribonuclease enzyme and, more particularly, Serratia
Mercenses.

[0017]The present invention suggests that cancer can be treated by
reducing circulating DNA levels. Circulating DNA levels are higher in the
blood of cancer patients than in healthy controls. Stroun discloses in
U.S. Pat. No. 5,952,170 a method of diagnosing cancers, wherein
extracellular DNA in the blood is used for diagnostics and for a
prognosis of a clinical course of a malignant disease. Hoon and Gocke
disclose in U.S. Pat. Nos. 6,465,177 and 6,156,504, respectively, a use
of blood's extracellular DNA to define mutations in oncogenes and
microsatellic fragments of genes. These patents also disclose usages of
blood's extracellular DNA for studying genome instability in tumors.

[0018]There is no systematic analysis of blood's extracellular DNA
spectrum and its biological role prior to this invention. A search of the
prior art reveals no published data concerning a research of blood's
extracellular DNA performed without a polymerase chain reaction ("PCR").
Polymerase chain reactions can pervert a pattern of blood's extracellular
DNA because of a specificity of primers which are used for amplification.
Until recently, a genetic analysis of extracellular blood DNA was mainly
carried out by PCR or by blot-hybridization and it was directed to a
study of changes in certain fragments of a genome, s.a., e.g.,
microsatellites and separate genes during a malignant process.

[0019]There is thus no available knowledge about a genetic repertoire of
blood's extracellular DNA in cancer patients, about a biological role of
that blood's extracellular DNA in oncopatology, and about the potential
therapeutic effects of a destruction, an inactivation or a treatment of
these diseases.

[0020]The blood's extracellular DNA in cancer patients contains a unique
quantitative and qualitative repertoire of genes and regulating genetic
elements which greatly differ from that of DNA in a healthy human genome.
In contract to intracellular DNA, extracellular DNA in cancer patients
mainly contains unique human genes, including genes which are involved in
a development of and a maintenance of malignant behavior in cancer cells.
Because blood's extracellular DNA contributes to malignant growth in
cancer patients, a destruction of, a modification of, or a binding of
blood's extracellular DNA is useful because it slows down that growth.
These interventions are very useful in independent therapy and they also
increase an effectiveness of traditional methods of treatment.

[0021]The aforesaid new characteristics of this invention are based on new
ideas about mechanisms of oncological diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]As set for below the invention has been explained by detailed
description of embodiments without references to drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0023]The inventive method is realized as followed:

1. Materials and Methods.

[0024]The following agents were used which destroys extracellular blood
DNA: bovine pancreatic DNAase (Sigma and Samson Med), recombinative human
DNAase 1 (Domase alpha; Genetech), Serratia Mercenses extracellular
nuclease. The solutions of DNA-se for administration was made by
dissolving of mother solutions of DNA-se in sterile phosphate buffer just
before administration.

[0025]Extracellular DNA from blood plasma was isolated as follows: fresh
plasma (no more than 3-4 hours after sampling) was centrifuged on
Ficoll-PlaquePlus (Amersham-Pharmacia) during 20 minutes at 1500 g at
room temperature. 1/2 of plasma was detached, not affecting the rest of
cells on the Ficoll pillow, and further centrifuged at 10000 g during 30
min for separation from cell fragments and debris. Supernatant was
detached, without affecting of the sediment, and was toped up to 1% of
sarkosil , 50 MM tris-HCl, pH 7,6, 20 MM EDTA, 400 MM NaCl, and than
mixed with equal volume of phenol-chloroform(1:1) mixture. The prepared
emulsion was incubated during 2 hours at t=65° C., then
phenol-chloroform mixture was separated by centrifuging (500 g during 20
minutes, room temperature).

[0026]The procedure of deproteinisation with phenol-chlorophorm mixture
was repeated 3 times, and then the water phase was processed with
chloroform and diethyl ether. Separation from organic solvents was made
by centrifugation at 5000 g during 15 minutes). Then equal volume of
izopropanol was added to resulting aqueous phase and the mixture was
incubated overnight at 0° C. After sedimentation the nucleic acids
were separated by centrifugation at 10000 g during 30 minutes. The
sediment of nucleic acids was dissolved in of 10 MM tris-HCl buffer, pH
7, 6 with 5 MM EDTA, and inflicted to the CsCl gradient (1 M, 2.5M, 5.7M)
in test-tube for rotor SW60Ti. The volume of DNA solution was 2 ml,
volume of each step of CsCl was 1 ml. Ultracentrifugation was conducted
in L80-80 (Beckman) centrifuge during 3 hours at 250000 g. DNA was
collected from the surface of each gradient step into fractions. These
fractions were dialyzed during 12 hours (t=4° C.). Presence of DNA
in fractions was determined by agar electrophoresis and DNA was
visualized by ethidium bromide staining. The amount of DNA was determined
with specrophotometer (Beckman DU70) in cuvet (100 mcl) at wavelength of
220-230 nm.

[0027]Mice Lewis lung carcinoma and Erlich carcinoma were used in
experiments. Cells were cultivated in RPMI-1640 medium with 10% calf
serum and 1% penicillin-streptomycin in atmosphere of 5% CO2.

[0028]For tumors inoculation in mice the cells were cultivated till
monolayer is formed, then detached with tripsin-EDTA buffer. The cells
were washed 3 times by centrifuging in phosphate buffer and then
resuspended up to 0.5*107/ml concentration in the same buffer. The
cell viability was determined with metylene blue staining in
haemocitometer. Cells suspensions with no less than 95% of living cell
were used for transplantation.

[0029]C57B1 mice and white randomly breeded mice from "Rappolovo" animal
house were used. Weight of animals was 24-26 g. 6-7animals were kept in
one cage on a standard diet without limitation of water. LLC cells in
dose 5*105 per mice in 0.1 ml of phosphate buffer were transplanted
into thigh soft tissues. Erlich tumors were transplanted by
administration of 0.2 ml of 10% cell suspension in physiological
solution.

[0030]In some experiments level of extracellular DNA in blood plasma was
quantified. DNA was isolated according to aforesaid protocol. The DNA
level was measured with PicoGreen kit. Electrophoresis of extracellular
blood DNA was performed with 1% agar gel. DNA was visualized with
etidium-bromide solution. The levels of high molecular DNA fraction (more
than 300 base pairs) were determined by densitometry. Lambda phage DNA,
digested with EcoRI and HindIII was used as electrophoresis marker.

EXAMPLE 1

Inhibition of Erlich Carcinoma Growth

[0031]Recombinant human DNAase 1 (Genentech) was used.

[0032]1 group: 10 mice bearing Erlich carcinoma was used as control. The
mice were injected with 0.2 ml of phosphate buffer intraperitoneally
twice a day every day from day 3 to day 7 after the tumor cell
transplantation.

[0033]2 group: 10 mice bearing Erlich carcinoma were introduced with
intraperitoneal injections of DNAase in dose of 1 mg/kg of body weight in
0.2 ml of phosphate buffer four times daily every day from day 3 to day 7
after the tumor cell transplantation.

[0034]3 group: 10 mice bearing Erlich carcinoma were administered with
intraperitoneal injections of DNAase in dose of 0.5 mg/kg of body weight
in 0.2 ml of phosphate buffer four times daily every day from day 3 to
day 7 after the tumor cell transplantation.

[0035]4 group: 10 mice bearing Erlich carcinoma were administered with
intraperitoneal injections of DNAase in dose of 0.1 mg/kg of body weight
in 0.2 ml of phosphate buffer four times daily every day from day 3 to
day 7 after the tumor cell transplantation.

[0036]5 group: 10 mice bearing Erlich carcinoma were administered with
intraperitoneal injections of DNAase in dose of 0.05 mg/kg of body weight
in 0.2 ml of phosphate buffer four times daily every day from day 3 to
day 7 after the tumor cell transplantation. The results were evaluated as
tumor Growth Inhibitory Index(GII) (%) at the last day of DNAase
injections. The quantification of blood plasma extracellular DNA and its
electrophoretic qualification were also performed.

[0039]The presented data demonstrated that sufficiently high doses of
DNAase 1 are needed to achieve the better therapeutic effect.

EXAMPLE 2

Inhibition of Erlich Carcinoma Growth

[0040]Recombinant human DNAase 1 (Genentech) was used.

[0041]5groups of mice bearing LLC were used.

[0042]1 group - 7 mice - the control.

[0043]2 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 1 mg/kg of body weight twice a day every day from
day 3 to day 5 after the tumor cell transplantation.

[0044]3 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 1 mg/kg of body weight twice a day every day from
day 3 to day 10 after the tumor cell transplantation.

[0045]4 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 1 mg/kg of body weight twice a day every day from
day 3 to day 15 after the tumor cell transplantation.

[0046]5 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 1 mg/kg of body weight twice a day every day from
day 3 to day 18 after the tumor cell transplantation.

[0047]6 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 1 mg/kg of body weight twice a day every day on
3,5,7,9,11,13,15 and 17 day after the tumor cell transplantation.

[0048]7 group - 6 mice were administered with intraperitoneal injections
of DNAase in dose of 0.5 mg/kg of body weight four times daily every day
from day 3 to day 10 after the tumor cell transplantation. The results
was evaluated as animal survival on day 30 and day 50 after the tumor
cell transplantation. The results are presented in the table 2.

[0049]Animal survival on day 30 and day 50 after the tumor cell
transplantation.

[0050]The presented data demonstrated that the therapy efficacy increases
as the treatment time extends. The therapy efficacy is decrease if it is
not uninterrupted. Multiple-dose administration is preferred.

EXAMPLE 3

Lung Carcinoma Treatment

[0051]54-years-old man has been admitted to the hospital with diagnosis of
lung carcinoma.

[0052]By patient's agreement, due to lack of any available treatment
modality, subcutaneous injections of domaze-alpha were prescribed. The
treatment began with administration of daily dose of 50 mkg/kg. Every
consecutive day blood extracellular DNA level was measured and blood
extracellular DNA was fractioned by electrophoresis. Once a week the
primary tumor site and metastases were checked with X-rays and
NMR-tomography. After initial 7 day period the domaze-alpha daily dose
has been increased up to 100 mkg/kg because of no changes in level and
electrophoresis pattern of blood extracellular DNA and no reactions from
primary site of the tumor and the metastases. Because of no changes after
another 7 days the dosing has been increased up to 150 mkg/kg. Two days
after the first injection of the preparation in dose 150 mkg/kg the
material recession (more than 50%) of the blood extracellular DNA
fraction with the size more than 300 base pairs has been observed
although total amount of extracellular DNA has not been greatly decreased
(less than 20%). During the next 4 days the patient's general condition
has noticeably improved and on day 7 of this cycle of therapy
25%--decreasing of primary tumor lesion size and signs of regression of
two bone metastases have been shown by NMR-scanning and X-ray
examination. The probes of patient's extracellular DNA taken before the
treatment started and 21 days after the beginning the therapy were cloned
by means a method which allowed to construct non amplificated plasmid
libraries of blood extracellular DNA with representativeness up to one
million of clones with the average size of 300-500 base pairs. The DNA
which have been isolated with aforesaid protocol was additionally
deproteinizated with proteinase K (Sigma) at t=65 C for the removing of
firm-binded proteins. After the deproteinization and single-stage
treatment of phenol-chloroform mixture (t=65° C.) DNA was
precipitated overnight with 2.5 volumes of ethanol. Then DNA was treated
by Eco RI restrictase during 3 hours or by Pfu polymerase (Stratagene) in
presence of 300 mkM o f all desoxynucleotidtriphosphates for sticky-ends
elimination. The completed DNA was phosphorylated by polynucleotidkinase
T4 (30 U, 2 h.). The preparations were ligated to pBluescript plasmide
(Stratagene), which has been digested with EcoRI or PvuII and
dephosphorylated by phosphatase CIP (Fermentas) during 1 hour. 1 mkg of
vector and 0.1-0.5 mkg of serum DNA were used. The process of ligation
was conducted with Rapid Legation Kit (Roche) during 10 hours at
T=16° C. The volume of this mixture was 50 mkl. The ligated
library was transformed into DH12S cells (Life Technologies) by meant of
electroporator E. Coli porator (BioRad). 12-20 electroporation covets
were used for the transformation of one library. The library serial
dilutions of 10-4, 10-5 and 10-6 were cloned on 1.5% agar
and LB media supplemented with 100 mkg/ml of ampicilline. In both cases
the libraries represented 2-3*106 clones.

[0053]Analysis of 96 randomly selected clones with the size 300-1000 base
pairs from the "before treatment" library showed that 55 from 96 clones
were the unique sequences of human DNA. For the 15 sequences from 55 the
gene function or corresponding gene product were identified with
HumanGeneBank.

[0054]Analysis of 100 clones selected randomly from the "21 day after
treatment" library showed that more than 90% sequences of clones
represented short fragments of repetitive DNA of human genome with
dominance of alpha-satellite DNA.

[0055]Hence the use of DNAase in doses which are sufficient for destroying
extracellular blood DNA with size higher than 300 base pairs leads to
disappearing of unique fragments of human genome from extracellular blood
DNA, including those involved into development and maintenance of cancer
cells malignant behavior. At the same time the tumor respond to applied
therapy.

EXAMPLE 4

The Treatment of Malignant Low Differentiated Lymphoma Invading the Spleen
and Portal Vien and Metastases in the Liver

[0056]49-years-old woman has been admitted to the hospital with the fever
(39 C), progressive jaundice, liver failure and being under suspicion of
acute hepatitis suffering. During the inspection malignant lymphoma with
the diffusely defeats of spleen and gates of liver and multiply
metastases in liver were revealed. By patient's agreement, due to the
lack of any specific treatment and because of progressing of the disease,
intravenous injections of bovine pancreatic DNAase were prescribed. Twice
a day measuring of level o f blood extracellular DNA and it's
electrophoretic fractioning were conducted. During the first day 500000
units of enzyme were administered as 2 6-hour infusions. Later this dose
was increased by 1 000 000 units per day. When the dose was 5500000 units
daily the 50% decrease of blood extracellular DNA and disappearance of
fraction of DNA with size more than 300 base pairs were noted. As the
continued DNA infusions at 5500000 units per day were being performed the
patient's general condition was being improved, fever and jaundice
disappeared, biochemical indexes of blood taken a turn to the better.
Control Doppler examination which has been made at day 20 after the
beginning of the treatment showed significant reduction (more than 40%)
of lesion in the spleen and disappearance of more than half of all
metastatic sites in the liver. The woman was moved to another hospital
for conducting chemotherapy.

[0057]Hence the use of DNAase in doses which are sufficient for destroying
extracellular DNA of blood with size higher than 300 base pairs leads to
tumor regression according to the inventive method.

EXAMPLE 5

The Study of Influence of Polyclonal Serum Containing the Antibodies
Against DNA on the Growth of Erlich Carcinoma of in Mice Treating with
DNAase

[0058]Antibodies against DNA were isolated from the blood of patients with
systemic lupus erythematosus according to method of Shuster. A. M.
(Shuster A. M. et. al., Science, v.256, 1992, pp.665-667). Such anti-DNA
antibodies could not only bind DNA but also hydrolyze it. Human
recombinant DNAase 1(Genetech) was used.

[0059]1 group- 7 mice bearing Erlich carcinoma - control

[0060]2 group- 6 mice bearing Erlich carcinoma and being have got
intravenous injection of human anti-DNA antibodies (Ig G) in dose of 200
mkg per animal on day 3 after the carcinoma transpalantation. Mice also
have been administered with DNAase in dose 0.5 mg/kg 4 times
intraperitonealy a day from day 3 to day 7 after the tumor
transplantation.

[0061]3 group - 6 mice bearing Erlich carcinoma and being have got
intravenous injection of human non-specific immunoglobulin (Ig G) in dose
of 200 mkg per animal on day 3 after the carcinoma transpalantation. Mice
also have been administered with DNAase in dose 0.5 mg/kg 4 times
intraperitonealy a day from day 3 to day 7 after the tumor
transplantation.

[0062]4 group- 6 mice bearing Erlich carcinoma and being have got
intraperitoneal treatment of human DNAase in dose of 0.5 mg/kg 4 times
intraperitonealy a day from day 3 to day 7 after the tumor
transplantation.

[0063]The effect was evaluated as the tumor growth inhibition on day 7
after the tumor cell transplantation (TGI, evaluated in percents). The
results are presented in the table 3.

[0065]The presented data demonstrated that the combined therapy with
DNAase and the agent binding blood exracellular DNA has more noticeable
antitumor effect.

EXAMPLE 6

The Study of Degradation Kinetics of High Molecular Weight Fraction (Size
More Than 300 Pairs of Bases) of Blood Extracellular DNA of Breast Cancer
Patient in Presence of Bovine Pancreatic DNAase, Proteinase K and Bovine
Pancreatic DNAase, Lipase and Bovine Pancreatic DNAase and Extracellular
Desoxyrybonuclease Serratia Mercenses, Which has Ribonuclease Activity
and is as Destroyed and Modificating Agent at the Same Time

[0066]The respective enzyme was added to a sample of patient's plasma and
incubated for 45 minutes at 37° C. 45 minutes later the reaction
has being stopped and isolation and electrophoretic fractioning with
densitometry of blood extracellular DNA have being performed.

[0069]The presented data demonstrated that the combined therapy with
DNAase and the agent modificating blood exracellular DNA binding with
proteins, lipids and ribonucleic acids leads to more effective
degradation of high molecular fraction (size more than 300 pairs of
bases) of blood extracellular DNA

EXAMPLE 7

The Study of the Influence of Different Methods of Destroying
Extracellular DNA on it's Pathogenic Properties

[0070]C57B1 mice have been inoculated with high metastatic or low
metastatic strain of LLC tumor. On the 9 th day after the inoculation
animals were euthanized and pool of their blood plasma was collected. The
summary fraction of extracellular blood plasma DNA was kept in phosphate
butler at t=-20° C.).

[0071]7 groups of mice inoculated with low metastatic strain of LLC were
included in the experiment.

[0072]1group- 6 mice grafted by low metastatic LLC strain.

[0073]2 group- 6 mice grafted by low metastatic LLC strain and were
subjected to twice repeated intravenous administration (on 7 and 8 day
after inoculation) of summary fraction of extracellular DNA from mice
grafted by high metastatic strain (before the administration 0.05 mkg of
DNA have been dissolved in 500 mkl of fresh heparinized blood)

[0074]3 group- 6 mice grafted by low metastatic LLC strain and were
subjected to twice repeated intravenous administration (on 7 and 8 day
after inoculation) of summary fraction of extracellular DNA from mice
grafted by high metastatic strain (before the administration 0.05 mkg of
DNA have been dissolved in 500 mkl of fresh heparinized blood). Before
the administration the sample with DNA has been disinfected
photochemically (by adding 1 mkM of methylen blue stain and exposure of
red light during 10 min (˜60 000 lux).

[0075]4 group - 6 mice grafted by low metastatic LLC strain and were
subjected to twice repeated intravenous administration (on 7 and 8 day
after inoculation) of summary fraction of extracellular DNA from mice
grafted by high metastatic strain (before the administration 0.05 mkg of
DNA have been dissolved in 500 mkl of fresh heparinized blood). Before
the administration the sample with DNA has been mixed with 10 mkg of
hydrolytic anti-DNA antibodies.

[0076]5 group - 6 mice grafted by low metastatic LLC strain and were
subjected to twice repeated intravenous administration (on 7 and 8 day
after inoculation) of summary fraction of extracellular DNA of mice graft
by high metastatic strain (before the administration 0.05 mkg of DNA have
been dissolved in 500 mkl of fresh heparinized blood). Before the
administration 1 mkg of the fragment A of the plant toxin Ricin was added
to the sample and the mixture was incubated during 1 hour at =37°
C. Ricin is the representative of RIP-toxins family (proteins
inactivating ribosomes) which widely used for immunotoxins' development.
In addition to its capability to inactivate ribosomes these proteins also
can deadenilate and hydrolyze DNA. To realize of the toxic effect the
unit A of the type II RIP toxin should be delivered into cell by unit B.
In absence of subunit B chain A is not toxic, however
polyadeninglicosidase activity of chain A can be used for destruction of
DNA circulating in blood.

[0077]6 group - 6 mice grafted by low metastatic LLC strain were subjected
to twice repeated intravenous administration (on 7 and 8 day after
inoculation) of summary fraction of extracellular DNA from mice grafted
by high metastatic strain (before the administration 0.05 mkg of DNA have
been dissolved in 500 mkl of fresh heparinized blood). The DNA sample was
enzymatically methylated before the administration. (I. Muiznieks et.
al., FEBS Letters, 1994,v. 344,pp.251-254).

[0079]8 group - 6 mice grafted by low metastatic LLC strain were subjected
to twice repeated intravenous administration (on 7 and 8 day after
inoculation) of summary fraction of extracellular DNA of mice grafted by
high metastatic strain (before the administration 0.05 mkg of DNA have
been dissolved in 500 mkl of fresh heparinized blood). The sample with
DNA was incubated with 200 ng/ml of domase alpha during 30 minutes at
T=37° C. before the administration.

[0080]The number of lung metastases (N cp) was evaluated on day 15 after
the inoculation.

[0081]The results are presented in the table 5.

[0082]The number of lung metastases on day 15 after the tumor inoculation
subject to the extracellular DNA destruction method.

Pilot Clinical Trials of DNAse Enzyme Monotherapy in Patients With
Advanced Cancer of Different Origin

[0084]The trials were performed in St. Petersburg St. Petersburg Academy
of Advanced Medical Education; Department of Thoracal Surgery. Totally 12
patients were included according to following inclusion criteria:

[0109]Day 12-21: 100 mg per infusion (1 020 000 Kunitz units per day)
[0110]The efficacy was assessed on day 30 after start of therapy. All
patients demonstrated stabilization of the disease. (Spiral CT scan;
RECIST criteria). All patients demonstrated significant increase in
Karnofsky performance score; some patients show shrinkage of metastatic
nodules. So, DNAse therapy is effective in treatment of malignant tumors
of different origin.

In Terms of Different DNASE Enzymes:

EXAMPLE 9

Inhibition of Growth of Human Tumors in Nude Mice Under Treatment With
Different High-Dose DNASE Enzymes

[0111]DNase IIβ DLAD is an enzyme that degrades DNA during lens cell
differentiation and was purchased from Abnova Corporation. DNase ILI is
member of deoxyribonuclease family showing high sequence similarity to
lysosomal DNase I (Abnova Corporation). TURBO® DNase is genetically
reengineered form of bovine DNase I for greater catalytic efficiency than
conventional DNase I at higher salt concentrations and lower DNA
concentrations. The enzyme was purchased from Ambion.

[0112]All experiments were performed in 6-8 week old female nu/nu mice.
Eighty eight nude mice were randomly divided into control and
experimental groups as follows:

[0113]COLO205 (Human colon cancer) and NCl-H82 (Human lung cancer) cells
(10-to-12 million) were injected s.c in the left flank of animals. Once a
palpable tumor was observed seven daily intramuscular injections of DNASE
enzyme or saline were given as indicated in the table above. The
anti-tumor activity following DNASE treatment was assessed by measuring
the tumor dimensions at the day following the day of last injection in
the control (PBS) and DNASE-treated groups. The apparent tumor volume was
calculated using the formula [tumor volume
(mm3)=(Length×Width2)/2]. The results of treatment
expressed as % % of tumor growth inhibition in DNASE-treated animals in
comparison with controls arc presented at the above table. Thus,
different DNASE enzymes posses anti-cancer activity at doses used.

[0114]The foregoing descriptions of specific embodiments of the present
invention have been presented for purposes of illustration and
description. They are neither intended to be exhaustive nor to limit the
invention to the precise forms disclosed and, obviously, many
modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the
principles of the invention and its practical application, to thereby
enable others skilled in the art to best utilize the invention and its
various embodiments with various modifications as are suited to the
particular use contemplated. It is intended that the scope of the
invention be defined by the Claims appended hereto and their equivalents.
Therefore, the scope of the invention is to be limited only by the
following claims.